1. Dr. Marian Maher Lecturer of Biochemistry and Molecular Biology
Blood clotting
Dr. Marian Maher
Lecturer of Biochemistry and Molecular Biology

2. Lecture 1

3. Definition
Coagulation is designed to rapidly stop bleeding from a damaged blood
vessel in order to maintain a constant blood volume (Hemostasis)

4. Phases Initial vasoconstriction.
Formation of a loose and temporary platelet aggregate.
Formation of a fibrin mesh that binds to the platelet aggregate, forming a more stable hemostatic plug or thrombus.
Partial or complete dissolution of the hemostatic plug or thrombus.

5. A) PLATELET PLUG FORMATION
Platelets adhere to exposed collagen of damaged endothelium, get activated, and aggregate to form a platelet plug

6. A. Adhesion
VWF binds to collagen, and platelets bind to VWF via a membrane receptor on the platelet surface.
Binding to VWF stops the forward movement of platelets.
Once adhered, platelets get activated.

7. B. Activation Thrombin is the most potent platelet activator.
Thrombin binds to and activates protease- activated receptors, on the surface of platelets resulting in activation of phospholipase C :
diacylglycerol (DAG)
inositol trisphosphate (IP3).

8. 1. Degranulation: vasoconstriction activation of additional platelets.
DAG activates protein kinase C, a key event for degranulation.
IP3 causes the release of Ca2+ (from dense granules). The Ca2+ activates phospholipase A2, which cleaves membrane phospholipids to release arachidonic acid, the substrate for the synthesis of thromboxane A2 (TXA2) in activated platelets by cyclooxygenase1 (COX-1) TXA2 causes
vasoconstriction
activation of additional platelets.
Degranulation also results in release of
Serotonin causes vasoconstriction.
ADP binds to GPCRs on the surface of platelets, activating additional platelets activating additional platelets.
Platelet-derived growth factor, VWF, FV, FXIII, and fibrinogen

9. 2. Shape change:
Discoidal spherical with pseudopod-like processes
Facilitate platelet–platelet and
platelet–surface interactions
Is initiated by the release of Ca2+
from dense granules. Ca2+ bound
to calmodulin mediates the activation
of myosin light-chain.

10. C. Aggregation
Structural changes in a surface receptor expose binding sites for fibrinogen.
a single fibrinogen able to bind two platelets.
The fibrinogen is converted to fibrin and cross-linked by XIIIa coming from both the blood and the platelets. Fibrin strengthens the platelet plug.

11. The exposure of phospholipid phosphatidylserine (PS) on the surface of activated platelets allows formation of the Xase complex (VIIIa, IXa, X, and Ca2+) with subsequent formation of Xa and generation of thrombin.

12. B) Fibrin Meshwork Formation
Involves two unique pathways that converge to form a common pathway

13. Gla-containing proteases
Clotting factors:
The factors are glycoproteins that are synthesized and secreted by the liver
II,VII,IX,X,XI,XII
serine proteases
II,VII,IX,X
Gla-containing proteases
III,V,VIII
accessory proteins

14. NB: I (fibrin) is neither a protease nor an accessory protein.
Ca2+ is IV.
There is no VI.
XIII is a transglutaminase

15. II, VII, IX, X, XI, and XII proteins are converted from an inactive form, or zymogen, to an active form by proteolytic cleavage in which the protein product of one activation reaction initiates many others. The active proteins are enzymes that function as serine proteases.

16. III, V, and VIII activation can be caused by a conformational change in the protein in the absence of proteolysis. Non proteolytic proteins play a role as accessory proteins (cofactors) in the pathways.

17. II, VII, IX, and X are serine protease contaning γ-carboxyglutamate (Gla) residues
γ-Carboxylation is a posttranslational modification in which glutamate is carboxlated at the γ carbon of the liver.

18. This carboxylation reaction requires a protein substrate, CO2, O2, γ-glutamyl carboxylase, and the hydroquinone form of vitamin K as a coenzyme

19. Role of phosphatidylserine and calcium:
Negatively charged phosphatidylserine:
PS on intracellular (cytosolic) face of the plasma
injury
membrane exposed on the surface of blood vessel and
activated platelets.
Calcium ions:
Ca2+ binds the negatively
charged (Gla) of
(II, VII, IX, and X)

20. Pathways: Extrinsic pathway:
(TF) or (III) is abundant in vascular subendothelium becomes exposed when blood vessels get injured. It is accessory protein not a protease.
TF is accessory protein not a protease. Any injury that exposesIII to blood rapidly (seconds) initiates the extrinsic (or TF) pathway.
TF binds a circulating Gla-containing protein, VII, activating it through conformational change. Binding of VII to TF requires the
presence of Ca2+ and phospholipids. (VII can be activated proteolytically by thrombin).
The TF–VIIa complex then binds and activates X by Proteolysis forming an Xase complex.
The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) that, in a Xa-dependent process, binds to the TF–VIIa complex and prevents further production of Xa.

21. Lab test for extrinsic pathways
through common pathway is [PT] using thromboplastin (Thromboplastin is a combination of phospholipids + III).

22. Intrinsic pathway: All factors involved are present in the blood
Contact phase: activation of XII to XIIa by conformational change through binding to a negative surface (XII can be activated proteolytically by thrombin).
Factor X–activation phase: XIIa activates XI (can be activated proteolytically by thrombin). XIa activates IX. IXa (a Gla-containing protein) combines with VIIIa The complex activates X (a Gla-containing serine protease).

23. Lab tests for intrinsic through common pathway [aPTT]
Lab tests for intrinsic through common pathway [aPTT]. (A derivative, partial thromboplastin, contains just the phospholipid portion because III isn’t needed to activate the intrinsic pathway).

25. All of the following are vit K dependent factors except:
MCQ
All of the following are vit K dependent factors except:
Factor VIII
Factor II
Factor X
Factor IX
Factor VII

26. Conversion of prothrombin to thrombin
3. Common pathway:
Conversion of prothrombin to thrombin
Xa associates with Va (a bloodborne accessory nonenzymic protein) and, in the presence of Ca2+ and phospholipids, forms a membrane-bound complex referred to as prothrombinase.
The complex cleaves prothrombin II to thrombin IIa. Cleavage excises the Gla-containing region, releasing thrombin from the membrane and, thereby, freeing it to activate fibrinogen I in the blood.
The peptide travels to the liver where it is thought to act as a signal for increased production of clotting proteins.

27. Conversion of fibrinogen to fibrin by thrombin:
The N termini of the Aα and Bβ chains form “tufts” on the central. The tufts are negatively charged and result in repulsion
between fibrinogen molecules.
Thrombin cleaves the
charged tufts
Fibrinogen becomes fibrin,
a soft (soluble) fibrin clot
is formed.

28. Cross-linking of fibrin:
XIIIa, a transglutaminase, (XIII is also activated by thrombin) covalently links the γ- carboxamide of a glutamine in one fibrin molecule to the ε-amino of a lysine in another forming an isopeptide bond and release of ammonia.

29. Fibrin polymer (soft clot)
This converts the soft clot to a hard (insoluble) clot.
Thrombin
Fibrin polymer (soft clot)
Fibrinogen
XIII a
Cross-linked fibrin
(Hard clot)

30. Lecture 2

31. C) Limiting Clotting & Clot Dissolution
The ability to limit clotting to areas of damage (anticoagulation) and to remove clots once repair processes are underway (fibrinolysis)

32. to limit their formation beyond the injured area.
Inactivating proteins
to limit their formation beyond the injured area.
Antithrombin:
Antithrombin III (ATIII or AT) is a hepatic protein that circulates in the blood.
It inactivates free thrombin by binding to it and carrying it to the liver, preventing it from participating in coagulation.

33. The affinity of ATIII for thrombin is greatly increased when ATIII is bound to heparin
ATIII also inactivates Xa , IXa, XIa, XIIa, and the VIIa–TF complex.

34. Protein C–protein S complex:
Thrombomodulin, an integral membrane glycoprotein of endothelial cells, binds thrombin, thereby decreasing thrombin’s affinity for fibrinogen and increasing its affinity for protein C.
Protein C, a circulating Gla-containing protein made in the liver, is activated by thrombin complexed with thrombomodulin.

35. Protein C in complex with protein S, also a Gla- containing protein, forms the activated protein C (APC) complex.
The APC cleaves the
accessory proteins Va and VIIIa
required activity of Xa .

36. Tissue factor pathway inhibitor (TFPI)
The extrinsic pathway is quickly inactivated by tissue factor pathway inhibitor (TFPI) binds to the TF–VIIa complex and prevents further production of Xa.

38. Fibrinolysis
Clots are temporary patches that must be removed once wound repair has begun.
Plasminogen, secreted by the liver into the circulation, binds to fibrin and is incorporated into clots. Activated to plasmin to cleave fibrin clot to fibrin-degradation products

39. Tissue plasminogen activator (TPA, t-PA), made by vascular endothelial cells and secreted in an inactive form in response to thrombin, becomes active when bound to fibrin–plasminogen.

40. Bound plasmin and TPA are protected from their inhibitors, α2-antiplasmin and plasminogen activator inhibitors, respectively.
Once the fibrin clot is dissolved, plasmin and TPA become available to their inhibitors.

41. Measurement of D-dimer, a fibrin-degradation product containing two cross-linked D domains released by the action of plasmin, can be used to assess the extent of clotting

42. Fibrinolysis system includes
Protein C
Plasminogen
Protein S
Antithrobin III
TFPI

43. Diseases: Factor XII deficiency:
A deficiency in XII does not lead to a bleeding disorder. This is because XI, the next protein in the cascade, can be activated proteolytically by thrombin.

44. Hemophilia:
Hemophilia is a coagulopathy, a defect in the ability to clot and an X-linked disorder.
Hemophilia A (most common, classical) results from deficiency of VIII
Hemophilia B results from deficiency of IX.
Hemophilia C results from deficiency of XI
The inactivation of the extrinsic pathway by TFPI results in dependence on the intrinsic pathway for continued production of Xa. This explains why individuals with hemophilia bleed even though they have an intact extrinsic pathway.

45. Von Willebrand disease
Deficiency of VWF results in von Willebrand disease (VWD), the most common inherited defect in the ability to clot (coagulopathy).

46. Glanzmann thrombasthenia
Rare defects in the platelet receptor for fibrinogen (decreased platelet function)
Autoantibodies to this receptor are a cause of immune thrombocytopenia (decreased platelet number).

47. Thrombophilia
Thrombophilia (hypercoagulability) causes a thrombus that forms in the deep veins of the leg (DVT) can cause a pulmonary embolism (PE) if the clot (or a piece of it) breaks off, travels to the lungs, and blocks circulation.
deficiencies of proteins C, S and ATIII
excess production of prothrombin.

48. presence of V Leiden (is a mutant form of V ) that is resistant to APC
presence of V Leiden (is a mutant form of V ) that is resistant to APC. It is the most common inherited cause of thrombophilia Women with FV Leiden are at even greater risk of thrombosis during pregnancy.
presence of antiphospholipid antibodies

49. Role of thrombin 1- Stimulates platelets aggregation
2- Activates factor VII, XII, XI also V, VIII
3- Activates fibrinogen
4- Activates factor XIII
5- Activates prothrombin (autoactivation)
6- Anticoagulant effect (delayed action) :
thrombin thrombomodulin complex activating protein C that cleaves Va and VIIIa
Activates plasminogen

50. Vitamin K dependent factors:
1-factor II 2-factor VII 3-factor IX 4-factor X 5-protein C 6-protein S

51. Inhibits vitamin k activation (II,VII,IX,X) Activates AT III and TFP I
Drugs
Therapeutic Anticoagulants:
Warfarin
Heparin
Plant source
Animal source
Source
In vivo only
Inhibits vitamin k activation (II,VII,IX,X)
In vivo and in vitro
Activates AT III and TFP I
Action
Oral tablets
Injections
Administration
Delayed hours
Instant
onset
Long
Short
Duration
Vitamin k injection
Protamine
Antidot

52. Therapeutic fibrinolysis
Urokinase
Is a plasminogen activator (PA) made in a variety of tissues.
recombinant (t-PA) has been widely used now.
Streptokinase
Also activates plasminogen and works on both free and fibrin-bound plasminogen.
Isolated from bacteria.

53. Antiplatelet drugs
Aspirin
Asprin irreversibly inhibits COX and consequently, TXA2 synthesis
ADP-receptor antagonists